A chromosomal reference genome sequence for the malaria mosquito, Anopheles moucheti, Evans, 1925

We present a genome assembly from an individual male Anopheles moucheti (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from a wild population in Cameroon. The genome sequence is 271 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.5 kilobases in length.


Background
Anopheles moucheti moucheti (hereafter An. moucheti) has the greatest geographical distribution within the Moucheti group, which includes An. bervoetsi and An.nigeriensis 1,2 .These species can only be distinguished by slight morphological characters 3 and / or using molecular tools 4,5 .An. moucheti is widely distributed across forested areas of West and Central Africa, and the persistence of this species is linked to its ability to lay eggs in lentic streams and rivers where its larvae develop preferentially and predominate over other malaria vectors 1,6 .An. moucheti females usually feed indoors and exhibit a preference for feeding on humans 7 although the species can also be found in forested areas of Gabon, away from any human presence 8,9 .An. moucheti is among the most important vectors of human malaria in the equatorial rain forest of Africa 1,10 .It is present in a large number of countries, from Guinea to Kenya 11 , and it has been found naturally infected with Plasmodium parasites across Central African countries, sustaining year-round transmission 1 .For instance, it can show infective biting rates up to 300 bites per person per year in villages located along slow moving rivers 7,10 .Moreover, An. moucheti has been recently incriminated for contributing to the origin of malaria in humans, being a potential bridge vector for malaria parasites from non-human primates to humans 8,9 .
The main genetic work carried out on An. moucheti has been focused on differentiation between members of the group.To this end, molecular markers have been developed that are based on the sequence variation in the ribosomal internal transcribed spacer ITS1 region 5 .Microsatellite loci have also been developed showing a low level of genetic differentiation among populations in Cameroon 5,12 .Several polymorphic inversions have been detected in An. moucheti, although their role in local adaptation or speciation is not yet understood 13 .Therefore, genomic characterization of this neglected but major malaria vector, An. moucheti, is critical for developing sustainable control strategies for malaria elimination in Central Africa.
The genome of the African malaria mosquito, Anopheles moucheti, was sequenced as part of the Anopheles Reference Genomes Project (PRJEB51690).Here we present a chromosomally complete genome sequence for Anopheles moucheti, based on a single male specimen from Ebogo, Cameroon.

Genome sequence report
The genome was sequenced from a single male Anopheles moucheti collected from Ebogo, Cameroon (3.384, 11.466).A total of 41-fold coverage in Pacific Biosciences singlemolecule HiFi long reads (N50 13.064 kb) were generated.
Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from a sibling male mosquito.Manual assembly curation corrected 48 missing joins or misjoins and removed 3 haplotypic duplications, reducing the scaffold number by 7.8% and reducing assembly size by 1.8%.
The final assembly has a total length of 271 Mb in 345 sequence scaffolds with a scaffold N50 of 92.649 Mb (Table 1).84.7% of the assembly sequence was assigned to three chromosomal-level scaffolds, representing two autosomes (numbered and oriented against the An.gambiae PEST strain assembly AgamP3 (14; GCF_000005575.2) with chromosome arms homologies for Myzomyia series taken from 15), and the X sex chromosome (Figure 1-Figure 6; Table 2).

BioSample ID ERS10527373
Isolate information male, whole organism

Assembly accession GCA_943734755
Accession      The assembly has a BUSCO 5.3.2 16completeness of 97.6% using the diptera_odb10 reference set.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
Anopheles moucheti offspring were reared from a wild caught gravid female collected from Ebogo, Cameroon (3.384, 11.466) by Sandrine Eveline Nsango.A single male idAnoMoucSN-F20_07 was used for Pacific BioSciences, a single sibling male idAnoMoucSN-F20_09 was used for Arima Hi-C.
For high molecular weight (HMW) DNA extraction one whole insect (idAnoMoucSN-F20_07) was disrupted by manual grinding with a blue plastic pestle in Qiagen MagAttract lysis buffer 17 and then extracted using the Qiagen MagAttract HMW DNA extraction kit with two minor modifications including halving volumes recommended by the manufacturer due to small sample size (Anopheles mosquitoes typically weigh 2-3 mg) and running two elution steps of 100 μl each to increase DNA yield.The quality of the DNA was evaluated using an Agilent FemtoPulse to ensure that most DNA molecules were larger than 30 kb, and preferably > 100 kb.In general, single mosquito extractions ranged in total estimated DNA yield from 200 ng to 800 ng, with an average yield of 500 ng.Low molecular weight DNA was removed using 0.8X AMpure XP purification.DNA was sheared to an average fragment size of 12-20 kb using a Diagenode Megaruptor 3 at speeds ranging from 27 to 30.Sheared DNA was purified using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.The concentration and quality of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer  For Hi-C data generation, a sibling male mosquito specimen (idAnoMoucSN-F20_09) was used as input material for the Arima V2 Kit according to the manufacturer's instructions for animal tissue.This approach of using a sibling was followed in order to enable all material from a single specimen to contribute to the PacBio data generation given we were not always able to meet the minimum suggested guidance of starting with > 300 ng of HMW DNA from a specimen.Samples proceeded to the Illumina library prep stage even if they were suboptimal (too little tissue) going into the Arima reaction.
To assist with annotation, which will be made available through VectorBase 18 in due course, RNA was extracted from separate whole unrelated lab-reared male and wild-caught female insect specimens (idAnoMoucSN-F4_01, idAnoMoucSN-F7_x) using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water, and its concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.Samples were not always ideally preserved for RNA, so qualities varied but all were sequenced anyway.

Sequencing
We prepared libraries as per the PacBio procedure and checklist for SMRTbell Libraries using Express TPK 2.0 with low DNA input.Every library was barcoded to support multiplexing.Final library yields ranged from 20 ng to 100 ng, representing only about 25% of the input sheared DNA.Libraries from two specimens were typically multiplexed on a single 8M SMRT Cell.Sequencing complexes were made using Sequencing Primer v4 and DNA Polymerase v2.0.Sequencing was carried out on the Sequel II system with 24-hour run time and 2-hour pre-extension.For Hi-C data generation, following the Arima Hi-C V2 reaction, samples were processed through Library Preparation using a NEB Next Ultra II DNA Library Prep Kit and sequenced aiming for 100x depth.RNA libraries were created using the directional NEB Ultra II stranded kit.Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (10X and Hi-C), or Illumina HiSeq 4000 (RNAseq) instruments.

Genome assembly
Assembly was carried out with Hifiasm 19 ; haplotypic duplications were identified and removed with purge_dups 20 .The assembly was then scaffolded with Hi-C data 21 using SALSA2 22 .
The assembly was checked for contamination as described previously 23 .Manual curation was performed using gEVAL 24 , HiGlass 25 and Pretext 26 .The mitochondrial genome was assembled using MitoHiFi 27 , which performs annotation using MitoFinder 28 .The genome was analysed and BUSCO scores were generated within the BlobToolKit environment 29 .Synteny analysis was performed with D-GENIES 30 and minimap2 31 .Table 3 contains a list of all software tool versions used, where appropriate.

Ethics/compliance issues
The genetic resources accessed and utilised under this project were done so in accordance with the UK ABS legislation (Nagoya Protocol (Compliance) (Amendment) (EU Exit) Regulations 2018 (SI 2018/1393)) and the national ABS legislation within the country of origin, where applicable.This data note by Nsango et al. present the methods and tools used for generating the first Anopheles moucheti genome assembly.Though An. moucheti is an important vector for human malaria, preferentially feeding indoor and on humans, and potentially acting as a bridge vector from non-human primates to humans, the authors explain genomic characterization of the species remains limited.Here, they used an individual male An.moucheti reared from a wildcaught gravid female for PacBio sequencing and a single sibling male for Arima Hi-C to construct three chromosomal pseudomolecules, the X sex chromosome, and the mitochondrial genome.They describe their methods from sample acquisition through genome assembly in appropriate detail to be replicated by others.
In Figure 3, the figure caption may be more descriptive if labeled "cumulative scaffold length" instead of "cumulative chromosome length" as only the dark blue "Arthropoda" line represents the chromosomes.Additionally, the figure legend truncates the "no-hit" line at around 225, which can be confusing as it is expected to end around 340. Moving the figure legend would be ideal.If this is not possible using BlobToolKit, setting the curve origin to "X" could be an improvement, as the "Arthropoda" line will originate where the "no-hit" line ends.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Vector Biology, Genomics
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Igor V Sharakhov
Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, USA The article by Nsango and co-authors present a new genome assembly obtained from an individual male Anopheles moucheti.The genome was sequenced using the PacBio HiFi procedure and scaffolded into three chromosomal pseudomolecules using Hi-C data obtained from a single sibling mosquito male.The complete mitochondrial genome was also assembled.The rationale for creating the Anopheles moucheti genome is clearly described as the species is an antropophilic malaria vector and a potential bridge vector for malaria parasites from non-human primates to humans.The manuscript provides sufficient details of methods and materials to allow replication by others.
It would be useful if chromosomes or chromosomal arms (if possible) are labeled in Figure 4. Have centromeric regions been captured either by reads or contigs?There are high frequency interaction spots outside the main diagonal in Figure 4.These could be indicative of polymorphic inversions.If indeed, this study assembled both haplotypes, then it is possible to test if they differ in the structural arrangements.This can be the case if the sequenced individual was a heterozygote.Also, building a Hi-C contact map for the second haply could answer this question.

Are the datasets clearly presented in a useable and accessible format? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Mosquito genetics, cytogenetics, genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Blob plot of base coverage in idAnoMoucSN-F20_07 PacBio HiFi reads against GC proportion for An.moucheti assembly idAnoMoucSN_F20_07.Chromosomes are coloured by phylum.Circles are sized in proportion to chromosome length.Histograms show the distribution of chromosome length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoMoucSN_F20_07/dataset/CALSEA01/blob.

Figure 1 .
Figure 1.Snail plot summary of assembly statistics for Anopheles moucheti assembly idAnoMoucSN_F20_07.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 271,332,225 bp assembly.The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (103,511,506 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 sequence lengths (92,649,126 and 283,162 bp), respectively.The pale grey spiral shows the cumulative sequence count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the diptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoMoucSN_F20_07/dataset/CALSEA01/snail.

Figure 3 .
Figure 3. Cumulative chromosome length for An.moucheti assembly idAnoMoucSN_F20_07.The grey line shows cumulative length for all chromosomes.Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule.The interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoMoucSN_F20_07/dataset/CALSEA01/cumulative.

Figure 4 .
Figure 4. Genome assembly of An. moucheti, idAnoMoucSN_F20_07: Hi-C contact map.Visualised in HiGlass.Chromosomes are arranged in size order from left to right and top to bottom.X chromosome signal is lower as individuals used for both PacBio and HiC are males.The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=GZ6DShODRcSMNgw7a0UIfQ.

Figure 5 .
Figure 5. Distribution plot of repeat masked sequence proportion against genomic position for chromosomes of An. moucheti, idAnoMoucSN_F20_07.1Mb windows with assembly gaps highlighted by larger blue circles, size is proportional to total size of gaps in the window (max 7800bp per window).

Reviewer Report 16
November 2023 https://doi.org/10.21956/wellcomeopenres.22437.r69866© 2023 Sharakhov I.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.